Parkinson's disease (PD) is a very common neurodegenerative disorder that affects more than 2% of the population over 65 years of age. PD is caused by a progressive degeneration and loss of dopamine (DA)-producing neurons, which leads to tremor, rigidity, and hypokinesia (abnormally decreased mobility). It is thought that PD may be the first disease to be amenable to treatment using stem cell transplantation. Factors that support this notion include the knowledge of the specific cell type (DA neurons) needed to relieve the symptoms of the disease. In addition, several laboratories have been successful in developing methods to induce embryonic stem cells to differentiate into cells with many of the functions of DA neurons.
While attempts have been made to propagate neural progenitor cells (partially differentiated precursors to neurons and glial cells) for use in neurotransplantation and for drug screening, these efforts have met with limited success. Neurobasal medium has allowed for fast doubling times of cultured neural progenitor cells, but these doubling times are observed for about one month, after which the cells differentiate and lose their progenitor phenotype. Typically, with the most optimal culture conditions, neural progenitor cells will survive for only about 10 passages in culture. In addition, only about 1-2% of neural progenitor cells survive cryopreservation. Moreover, current efforts to maintain neural progenitor cells in vitro require the use of a feeder layer and/or introduce animal components. Even with use of a feeder layer, neural progenitor cells have been maintained for only about 6 months. For clinical applications, it is desirable to obtain and maintain human neural progenitor cells that are free of animal components and do not require the use of a feeder layer.
Likewise, the development of stem cells for transplantation has been approached with an assumption that such pluripotent cells would have to be genetically modified in order to express the desired phenotype for therapeutic benefit, such as the neurotransmitter dopamine for treatment of Parkinson's disease. Alternatively, it has been assumed that one must expose the stem cells to conditions that would induce predifferentiation to achieve the desired phenotype.
There remains a need for a large quantities of undifferentiated neural progenitor cells and pluripotent or totipotent stem cells for transplantation and for drug screening, particularly for human progenitor and stem cells. A need also exists for cells that are capable of long-term proliferation in vitro. In particular, there is a need for methods of maintaining and propagating neural progenitor and pluripotent cells for extended periods of time, and for methods that optimize yield following cryopreservation.